Cancer is one of the leading causes of death worldwide, surpassing heart disease. 8.2 million people of the global population died from cancer in 2012 (WHO). Classical anti-cancer therapies for example, radiotherapy, chemotherapy and conventional surgical procedures, often suffer from poor selectivity and, thus, from severe toxic side effects to healthy tissue. Novel forms of treatment consist in the targeted delivery of bioactive molecules (drugs, cytokines, radionuclides, etc.) to the tumor environment by means of binding molecules specific to tumor-associated antigens. This will allow the selective direction of drugs towards target-positive tumor tissue and effectively kill malignant cells without harming healthy cells. This goes along with the development of so-called companion diagnostics enabling the determination of target positive tumors within a patient in order to in advance guarantee a rationally tailored strategy for individual cancer therapy. In this, the application of target specific imaging techniques has become an important diagnostic step revealing an impressive advancement during the last decades. Imaging techniques can provide critical information about presence and quantity of tumor-associated proteins, localization, early detection, distribution, patient stratification, and treatment monitoring (Stern, Case et al. 2013).
A crucial step towards tailored personalized anticancer therapies is the identification of selectively tumor-associated marker proteins. The serine protease Seprase (Fap-alpha; fibroblast activation protein alpha) is selectively overexpressed in cancer-associated fibroblast (CAFs) in more than 90% of human primary epithelial tumors such as breast, lung and colorectal cancers with little to no expression in normal fibroblasts or other normal tissues (Rui Liu 2012), making it an attractive target for cancer therapy and diagnosis. Seprase is a 170 kDa type II transmembrane cell surface protein belonging to the post-proline dipeptidyl aminopeptidase family. It is anchored in the plasma membrane by a short transmembrane domain, intracellularly exposing an amino terminal sequence, whereas a catalytic domain with a carboxyl-terminus remains in the extracellular space (Goldstein, Ghersi et al. 1997, Pineiro-Sanchez, Goldstein et al. 1997). The exact role of Seprase in tumor growth and invasion, the molecular mechanism(s) the enzyme is involved in as well as its natural ligands or substrates remain largely unknown.
Being Seprase a selective marker for tumor tissue a number of potential therapeutic strategies targeting said protein can be envisioned. The use of Seprase binding molecules in a number of different cancer models in vivo has shown that it is possible to efficiently impair tumor progression in a preclinical approach (Loeffler, Kruger et al. 2006, Ostermann, Garin-Chesa et al. 2008, Liao, Luo et al. 2009, Kraman, Bambrough et al. 2010, Wen, Wang et al. 2010). In contrast to that, targeting Seprase in a clinical setup of human cancer patients using the monoclonal antibody F19, its humanized version Sibrotuzumab (Welt, Divgi et al. 1994, Hofheinz, al-Batran et al. 2003, Scott, Wiseman et al. 2003), or the Seprase enzyme-inhibitor Talabostat (Narra, Mullins et al. 2007, Eager, Cunningham et al. 2009, Eager, Cunningham et al. 2009), has demonstrated only modest clinical efficacy. Interestingly, no significant toxicities were reported in the preclinical studies targeting Seprase (Welt, Divgi et al. 1994, Lee, Fassnacht et al. 2005, Loeffler, Kruger et al. 2006, Ostermann, Garin-Chesa et al. 2008, Liao, Luo et al. 2009, Santos, Jung et al. 2009, Kraman, Bambrough et al. 2010, Wen, Wang et al. 2010) although an expression also by multipotent bone marrow stem cells (BMSCs) is currently discussed. In summary, the favorable biodistribution of the Seprase-specific antibodies and the selective uptake in sites of metastatic disease in patient reported so far (Welt, Divgi et al. 1994, Scott, Wiseman et al. 2003) qualify Seprase as an attractive candidate for tumor targeting approaches. Due to the fact that it remains unknown if Seprase acts as a tumor suppressor (Wesley, Albino et al. 1999, Ramirez-Montagut, Blachere et al. 2004) or promotes tumor growth (Cheng, Dunbrack et al. 2002, Goodman, Rozypal et al. 2003, Huang, Wang et al. 2004) it might be favorable to develop highly selective ligands to Seprase for imaging techniques having no impact on function but providing information about localization, early detection, distribution, patient stratification, and treatment monitoring (Stem, Case et al. 2013).
For targeting of poorly vascularized tumors, the large size of antibodies and even their fragments might slow the rate of tissue penetration and by this hamper efficient delivery. Moreover, because of the extended blood circulation of antibodies they seem not optimal for diagnostic use especially in the context of imaging concepts. In addition to the foresaid, the molecular architecture of antibodies, with complex glycosylation pattern and disulfide bridges, requires complex cost-intensive manufacturing and complicates further functionalization e.g. by means of an imaging tracer. To overcome these limitations, as an alternative to antibodies so-called protein scaffolds have emerged during the last decades: Scaffolds provide a robust structural framework to precisely engineer interaction molecules tailored for the tight and specific recognition of a given target (Weidle, Auer et al. 2013). Most of them fold properly under non-reducing conditions and can be expressed in bacteria without the need for denaturation and refolding. Even chemical synthesis is an option for the production of some of the formats. Finally, they are well-suited for further functionalization (labelling, oligomerization, fusion with other peptides, etc.) to generate multi-functional binding molecules. Among the different scaffold-based approaches cystine-knot miniproteins (“knottins”) have shown great potential for the development of targeted diagnostics and therapeutics agents. For example, Cochran and co-workers generated radiolabeled miniproteins, 18F-FP-2.5D and 18F-FP-2.5F, for integrin-specific PET imaging of U87MG tumors, marked by good contrast, fast tumor targeting, rapid clearance from the body and relatively low uptake in normal tissues (Kimura, Cheng et al. 2009, Kimura, Levin et al. 2009, Kimura, Miao et al. 2010, Kimura, Jones et al. 2011, Liu, Liu et al. 2011). Miniproteins are small, 30-50 amino acid polypeptides containing three disulfide bonds that form the eponymous knotted structure (Kolmar 2009, Moore and Cochran 2012). The pseudoknot cystine topology is responsible for an extraordinary thermal, proteolytic and chemical stability, which is desirable for in vivo biomedical applications (Kolmar 2011). For example, without losing structural and functional integrity, miniproteins can be boiled in alkaline or acidic environment (Weidle, Auer et al. 2013). The disulfide-constrained loop regions tolerate broad sequence diversity, providing a robust molecular framework for engineering proteins that recognize a variety of biomedical targets.
There is a need in the art for Seprase binding molecules which are useful in diagnostic and therapeutic approaches for tumors expressing Seprase.
Seprase binding agents such as Seprase binding peptides are described herein which show high specificity and selectivity for human Seprase. The Seprase binding agents described herein are excellent tools for diagnostic applications, particularly for tumor imaging, and therapeutic applications by efficient targeting of the tumor microenvironment.